Influence of dietary L‐carnitine and lysine–methionine levels on reproductive performance and blood metabolic constituents of breeder ducks

Abstract The present study aimed to evaluate the influence of dietary supplementation of different levels of L‐carnitine and/or lysine–methionine (Lys‐Met) on reproductive performance of breeder ducks. Three L‐carnitine (0, 75 and 150 mg/kg) and three lysine–methionine (100%, 110% and 120% above the NRC (Nutrient requirements of poultry, 1994, National Academy Press) recommendations) levels were fed to 180 breeder ducks (144 females and 36 males) in a completely randomized design for 49 days. Laying performance and reproductive traits were evaluated; additionally, uric acid, total protein total, triglycerides, total cholesterol, low‐density lipoprotein, high‐density lipoprotein, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were assessed. The Lys‐Met above 100% NRC (Nutrient requirements of poultry, 1994, National Academy Press) recommendations with or without L‐carnitine improved feed utilization (p < .05). Furthermore, Lys‐Met above 100% recommendations without L‐carnitine improved egg fertility and hatchability. Fertility and hatchability improved in breeders fed on L‐carnitine with 120% Lys‐Met (p < .05). Serum glucose increased and total cholesterol reduced on 100% Ly‐Met without L‐carnitine or 110% Ly‐Met with 150 mg L‐carnitine (p < .05). Glucose was reduced, while total cholesterol increased on 75 mg L‐carnitine and 100% Lys‐Met (p < .05). Increasing Lys‐Met without L‐carnitine reduced serum protein (p < .05). Albumin and ALT increased on 75 mg L‐carnitine–100% Lys‐Met and reduced on 150 mg L‐carnitine–120% Lys‐Met (p < .05). There were no interaction effects on globulin, uric acid and AST (p > .05). Thus, based on findings, breeder ducks responded to dietary Lys‐Met more efficiently than L‐carnitine; however, more research is needed to evaluate also economic aspects related to L‐carnitine dietary supplementation.

Moreover, Grisoni et al. (1991) and Attia (2003) reported significant reductions in abdominal fat and increased lean meat deposition with lysine supplementation in broiler chickens and ducks, respectively.
These amino acids act mainly by suppressing lipogenesis through inactivation of fatty acid synthase and promotion of lipolysis by stimulating the secretion of hormone-sensitive lipase (Alagawany et al., 2016;Reda et al., 2015;Takahashi & Akiba, 1995).
L-carnitine is the biological active form of carnitine, synthesized endogenously from lysine and methionine. L-carnitine regulates lipid and energy metabolism (Rehman, Chand, et al., 2017;Rehman, Naz, et al., 2017) by promoting mitochondrial β-oxidation of long-chain fatty acids (Abd El-Wahab et al., 2015). Studies have shown that exogenous carnitine reduces the concentration of plasma, very-lowdensity lipoprotein cholesterol (VLDL-C) and VLDL triglycerides (TG) in hyperlipidaemic rabbits and plasma lipoprotein levels in type 2 diabetic patients with hypercholesterolaemia (Rajasekar et al., 2005;Rehman et al., 2018). Plasma glucose concentration increased with carnitine supplementation due to increased fatty acid oxidation and subsequent decreased oxidation of gluconeogenic precursors (Greenwood et al., 2001). However, poultry have limited ability to synthesize carnitine in diets low in lysine and methionine. This has increased research interest in L-carnitine supplementation of poultry diets. Supplementing diets with L-carnitine was found to reduce carcass fat deposition in ducks (Arslan, 2006), but available reports on the effects on reproductive performance and blood metabolites of this species are still limited. Therefore, the present study investigated the effects of different dietary levels of L-carnitine and lysine-methionine (Lys-Met) on some reproductive traits and blood constituents of native breeder ducks.

| MATERIAL S AND ME THODS
All the experimental procedures were carried out according to the approved protocols by the Institutional Animal Care and Use Committee (IACUC) of the Institutions, and care was followed to minimize the number of animals used.
Thus, based on findings, breeder ducks responded to dietary Lys-Met more efficiently than L-carnitine; however, more research is needed to evaluate also economic aspects related to L-carnitine dietary supplementation.

K E Y W O R D S
carnitine, duck, essential amino acids, reproduction, serum metabolites Feed and water were provided ad libitum during the experimental period. The lighting programme was 16 hr of light and 8 hr of dark throughout the duration of the study.

| Laying performance and reproductive traits
Feed intake was monitored per pen as the difference between the quantities of fed and leftover. Eggs were recorded per pen, and percentage egg production was calculated as 100 × (eggs collected ÷ hens in the pen). All eggs produced were weighed using a digital portable scale (Jadever JKH-500 series; SmartFox) sensitive to ±0.1 g, and the mean egg weight was recorded per pen. Feed conversion ratio (FCR) was calculated as g feed consumed to g egg produced.
Eggs collected were labelled per replicate and stored at 17°C and 74% relative humidity for 1 week. Eggs (68.7 ± 1.8 g) were candled and fertile ones incubated in the setter at 37.5°C and 60% humidity with regular turning for the first 25 days. On Day 25, eggs were transferred to the hatcher operated at 37°C and 71% relative humidity. At the end of 28 days of incubation, ducklings were removed and counted. Fertility and hatchability parameters were calculated as:

| Blood biochemical constituents
On Day 49 of the trial, three representative birds per replicate were selected and blood samples (1 ml/bird) collected, from the wing vein, for biochemical analysis according to Jahanpour et al. (2013). Blood samples were transferred from the syringe into sample tubes coated with 10 mg of ethylenediaminetetraacetic acid (EDTA) and centrifuged at 1000 g × 20 min to ensure separation of the blood cells from the plasma. Plasma constituents were analysed following the standard protocols using the Roche Cobas Integra 400 Plus autoanalyzer (Roche Diagnostics GmbH). Uric acid, total protein total, TG, cholesterol, low-density lipoprotein (LDL), high-density lipoprotein (HDL), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were assayed using commercial kits (Teif Azmoon Pars, Co.).

| Statistical analysis
Data were analysed using the GLM procedure of SAS software (SAS, 2000). The model included L-carnitine and Lys-Met as main effects. The interaction among main effects was included in the model. Means were compared using Duncan's multiple range test and significance levels reported at 5% probability. The model used was as follows: Y ijk = μ + A i + B j + AB ij + e ijk , where μ = the common mean, A i = the effects of the L-carnitine, B j = the effect of the Lys-Met, AB ij = the effect of the ith A with the jth B, and e ijk = the random error. Before performing the statistical analysis, all data were tested by the normality test and transformed, if necessary.

| Laying performance and reproductive traits
The results of laying performance and reproductive traits of the ducks ( Table 2) showed no interaction effects on feed intake, henday production, egg weight and egg mass of the ducks (p > .05).
Dietary Lys-Met above 100% of NRC (1994) recommendations with or without L-carnitine improved FCR (feed: gain) of ducks (p < .05). In the main effects, neither L-carnitine nor Lys-Met level affected the performance parameters observed (p > .05).
Egg fertility and hatchability indexes were significantly influenced by Lys-Met and Lys-Met interaction (p < .05). Feeding of Lys-Met above 100% of NRC recommendations without L-carnitine improved egg fertility and hatchability, but the addition of L-carnitine (75 or 150 mg) improved fertility and hatchability only when Lys-Met was increased to 120% of the NRC recommendations (p < .05).  Based on the main effects, L-carnitine addition did not affect the reproductive parameters (p > .05), but these were improved with increasing Lys-Met above 100% of NRC recommendations (p < .05).

| Metabolic parameters
The results of blood energy and protein metabolism parameters are presented in Tables 3 and 4 Leibetseder (1995) also found no effect of dietary L-carnitine (500 mg/kg) on laying performance of broiler breeders.
These data might imply that the effects of L-carnitine and Lys-Met are additive rather than antagonistic. Both Lys and Met are essential for the synthesis of egg and body protein, and a shortage in either of these amino acids leads to a reduction in egg production (Hiramoto et al., 1990). L-carnitine, which is mainly biosynthesized from Lys and Met (Abd El-Wahab et al., 2015;Arslan, 2006), regulates βoxidation of fatty acids and prevents oxidative reactions by forming a defence line against reactive oxygen species (Arenas et al., 1998).
A sufficient supply of L-carnitine in the diet is therefore essential to provide for these functions and preserve Lys and Met from conversion to carnitine, making them available for tissue synthesis.
Supplementation of low protein diets with carnitine in broiler chickens did promote growth in animals through its methionine-sparing effect (Golrokh et al., 2016;Panahi et al., 2019;Tufarelli et al., 2020). Owen et al. (1996) also reported sparing of branched-chain amino acids from oxidation in tissues with an increased supply of carnitine.
Improving egg fertility and hatchability can be achieved by increasing Lys-Met alone above 100% of NRC recommendations or by the addition of L-carnitine. This pattern further confirms the sparing effect of these amino acids from endogenous biosynthesis of carnitine. The beneficial effects of L-carnitine on poultry fertility are documented. L-carnitine increases sperm quantity and quality by protecting sperm against per-oxidative damage and dysfunction (Alvarez & Storey, 1989). Active transporters in the epididymal lumen release circulating L-carnitine into the lumen, which is controlled by androgens and delivered into spermatozoa, where it remains (Cooper, Gudermann, et al., 1986;Enomoto et al., 2002;Jeulin & Lewin, 1996;Kobayashi et al., 2005).

| Metabolic parameters
We could not explain the pattern of energy and protein metabolism in this study, but a possible increase in fatty acid oxidation and the subsequently reduced oxidation of gluconeogenic precursors (Greenwood et al., 2001) could be a possible speculation.
The available findings on Lys and Met supplementation alone or in combination with L-carnitine on serum biochemical constituents in poultry are inconsistent. In opposition to our results, Arslan et al. (2004), Taraz and Dastar (2008) and Babak et al. (2015) found  (Ali et al., 2007) may also be implicated in the differences in serum composition observed between this study and those found in other poultry species.

| CON CLUS ION
It appears from the obtained findings that breeding ducks respond to Lys-Met more efficiently than L-carnitine. Increasing dietary Lys-Met above NRC recommendations has no effect on egg production but improved feed utilization, egg fertility and hatchability. The pattern of the hens' response in terms of serum metabolites is not consistent, and this needs further investigations. Because carnitine is biosynthesized endogenously from Lys-Met, the supply of Lcarnitine in the diet will spare these essential amino acids. However, L-carnitine is still a relatively expensive additive in many countries and there is a need to validate the cost-effectiveness of its dietary TA B L E 4 Protein metabolic parameters and hepatic functionality of ducks fed different levels of dietary L-carnitine and lysine-methionine inclusion. However, their availability, ease of use and their positive impact on ducks may make their use in duck feeds justified.

ACK N OWLED G EM ENT
The authors gratefully acknowledge the financial support from Rasht Branch, Islamic Azad University (grant number 17. 16.4.18418). Open Access funding provided by Universita degli Studi di Bari Aldo Moro within the CRUI-CARE Agreement.

CO N FLI C T O F I NTE R E S T
None of the authors have any conflict of interest to declare.

DATA AVA I L A B I L I T Y
The data that support the findings of this study are available upon request from the corresponding author.